I always get pretty bad band distortion on restriction digests using BstYI or NdeI. The common factor is the CutSmart buffer. This doesn't happen to me with other enzymes. On the occasion when I cut with this buffer and extracted and precipitated the DNA the bands looked very nice, but this process is not always an option. Plus it doesn't make sense that NEB would have us use this buffer with so many enzymes if it messes up your gels. Still, I'm puzzled. Any ideas?
We routinely use NEB cutsmart buffer in combination with various enzymes like BamHI-HF , EcoRI-HF and NotI-HF to digest plasmids as well as bacmids. So far we did not come across any distortion or anomaly in the agarose gel running pattern. If you think using this this buffer distort your band, I would suggest to heat inactivate the enzyme reaction at 70C for 5-10 minutes before running it on the gel. Hope this helps.
Additionally you could also try digesting with BstYI or NdeI in NEB buffer 4 or NEB 2 or 2.1 respectively and compare whether these digestions makes any distortions.
Also make sure your DNA preparation is ultra pure.
Thanks for the suggestion on the buffers. What I've been doing now is not diluting the digest 1/2 with TE before adding loading juice. Seems to work. Thanks!
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques